Conventionally, the single molecule fluorescence detection has been performed, which uses an evanescent field generated over the surface of a transparent sample substrate by illuminating the sample substrate with an excitation light output from an excitation light source and causing the excitation light to be totally-reflected inside the sample substrate.
For example, in Non Patent Literature 1, in order to generate an evanescent field in the single molecule fluorescence detection, a configuration is employed in which a flat face of a prism and the sample substrate are deployed in such a manner that they are in parallel and facing with each other and the spacing therebetween is filled with a matching solution to match the refractive indices of both of them.
Also, in Non Patent Literature 2, the monomolecular-level DNA sequencing is performed using the total-internal reflection illumination scheme. 532-nm-wavelength and 635-nm-wavelength lasers are utilized for the fluorescence detections of fluorophore Cy3 and fluorophore Cy5, respectively. Taking advantage of biotin-avidin protein binding, a single target DNA molecule is immobilized onto the sample substrate that is filled with the solution. Then, a primer, which is labeled with the one Cy3 molecule, is introduced into the solution by exchanging the solution so that its concentration becomes constant and a single fluorescently labeled primer molecule is hybridized with the target DNA molecule. At this time, the Cy3 exists in the evanescent field and the binding position of the target DNA molecule is confirmed based on the fluorescence detection. After the Cy3 is photobleached by irradiating the high-power 532-nm excitation light, thereby suppressing the fluorescence light emission thereinafter. Next, polymerase and a dNTP (N is any one of A, C, G, and T) equipped with one type of base, which is labeled with the one Cy5 molecule, are introduced into the solution by performing the solution exchange so that their concentrations become equal to constant values, respectively. As a result of this introduction, as long as the dNTP is in the complementary relationship with the target DNA molecule, a fluorescently labeled dNTP molecule is captured into the elongated strand of the primer molecule. At this time, the Cy5 molecule exists in the evanescent field and the complementary relationship can be confirmed based on the fluorescence detection at the binding position of the target DNA molecule. After the Cy5 is photobleach by irradiating the high-power 635-nm excitation light, thereby suppressing the fluorescence light emission thereinafter. The above-described dNTP-capturing reaction process is repeated sequentially in a step-wise manner with the type of the base such as, for example, A→C→G→T→A→ (step-wise elongation reaction) to determine a sequence of bases in the complementary relationship with the target DNA molecule. Also, a plurality of target DNA molecules are immobilized within a single field-of-view of a fluorescence-detected image and the above-described dNTP-capturing reaction process is processed in parallel so that the simultaneous DNA sequencing of the plurality of target DNA molecules can be implemented. It is expected that the number of the simultaneous parallel processings at this time can be made dramatically larger as compared with the case of the conventional capillary-electrophoresis-based DNA sequencing.
As a method of generating the evanescent field over the sample substrate, as described in Non Patent Literature 3, there also exists the following method. Namely, both ends of the sample substrate are machined to form oblique planes thereon and the laser light is introduced from the oblique plane formed. The laser light propagates by taking advantage of the multiple reflection inside the sample substrate and the sample-immobilized area is illuminated.